The liver-specific microRNA-122 (miR-122) recognizes two conserved sites at the 5′ end of the hepatitis C virus (HCV) genome and contributes to stability, translation, and replication of the viral RNA. We show that stimulation of the HCV internal ribosome entry site (IRES) by miR-122 is essential for efficient viral replication. The mechanism relies on a dual function of the 5′ terminal sequence in the complementary positive (translation) and negative strand (replication), requiring different secondary structures. Predictions and experimental evidence argue for several alternative folds involving the miR-binding region (MBR) adjacent to the IRES and interfering with its function. Mutations in the MBR, designed to suppress these dysfunctional structures indeed stimulate translation independently of miR-122. Conversely, MBR mutants favoring alternative folds show impaired IRES activity. Our results therefore suggest that miR-122 binding assists the folding of a functional IRES in an RNA chaperone-like manner by suppressing energetically favorable alternative secondary structures.

The interplay between keratinocytes and immune cells, especially T cells, plays an important role in the pathogenesis of chronic inflammatory skin diseases. During psoriasis, keratinocytes attract T cells by releasing chemokines, while skin-infiltrating selfreactive T cells secrete proinflammatory cytokines, e.g., IFNγ and IL-17A, that cause epidermal hyperplasia. Similarly, in chronic graftversus-host disease, allogenic IFNγ-producing Th1/Tc1 and IL-17-producing Th17/Tc17 cells are recruited by keratinocyte-derived chemokines and accumulate in the skin. However, whether keratinocytes act as nonprofessional antigen-presenting cells to directly activate naive human T cells in the epidermis remains unknown. Here, we demonstrate that under proinflammatory conditions, primary human keratinocytes indeed activate naive human T cells. This activation required cell contact and costimulatory signaling via CD58/CD2 and CD54/LFA-1. Naive T cells costimulated by keratinocytes selectively differentiated into Th1 and Th17 cells. In particular, keratinocyte-initiated Th1 differentiation was dependent on costimulation through CD58/CD2. The latter molecule initiated STAT1 signaling and IFNγ production in T cells. Costimulation of T cells by keratinocytes resulting in Th1 and Th17 differentiation represents a new explanation for the local enrichment of Th1 and Th17 cells in the skin of patients with a chronic inflammatory skin disease. Consequently, local interference with T cell-keratinocyte interactions may represent a novel strategy for the treatment of Th1 and Th17 cell-driven skin diseases.In addition, in chronic graft-versus-host disease (GVHD), a major complication of allogenic stem cell transplantation, the KCmediated secretion of chemokines (CXCL9 and CXCL10) leads to the recruitment of alloreactive T cells into the skin. 14 These allogenic T cells predominantly belong to the IFNγ-producing Th1/ Tc1 and IL-17-producing Th17/Tc17 subpopulations and cause cutaneous manifestations, e.g., follicular erythema. [15][16][17] Although the pivotal role of KCs in non-contact-mediated communication during chronic skin inflammation is quite well understood, the direct interaction between KCs and T cells remains elusive. In particular, the potential of KCs to act as nonprofessional APCs, enabling them to costimulate T cells directly in the skin, is still debated.T cells require two distinct signals for activation and clonal expansion. The first signal is transmitted by the antigen-specific T cell receptor (TCR) on T cells, following recognition of antigenic peptides loaded on MHC class I or class II molecules expressed by APCs. The first signal secures the antigen specificity of the immune reaction. The second signal is transmitted through costimulatory receptors, dictating the progression to T cell activation. Between

Several antitumor therapies work by increasing reactive oxygen species (ROS) within the tumor micromilieu. Here, we reveal that L-plastin (LPL), an established tumor marker, is reversibly regulated by ROS-induced thiol oxidation on Cys101, which forms a disulfide bridge with Cys42. LPL reduction is mediated by the Thioredoxin1 (TRX1) system, as shown by TRX1 trapping, TRX1 knockdown and blockade of Thioredoxin1 reductase (TRXR1) with auranofin. LPL oxidation diminishes its actin-bundling capacity. Ratiometric imaging using an LPL-roGFP-Orp1 fusion protein and a dimedone-based proximity ligation assay (PLA) reveal that LPL oxidation occurs primarily in actin-based cellular extrusions and strongly inhibits cell spreading and filopodial extension formation in tumor cells. This effect is accompanied by decreased tumor cell migration, invasion and extracellular matrix (ECM) degradation. Since LPL oxidation occurs following treatment of tumors with auranofin or γ-irradiation, it may be a molecular mechanism contributing to the effectiveness of tumor treatment with redox-altering therapies.

MGMT promoter methylation status is currently the only established molecular prognosticator in IDH wild-type glioblastoma multiforme (GBM). Therefore, we aimed to discover novel therapy-associated epigenetic biomarkers. After enrichment for hypermethylated fractions using methyl-CpG-immunoprecipitation (MCIp), we performed global DNA methylation profiling for 14 long-term (LTS; >36 months) and 15 short-term (STS; 6-10 months) surviving GBM patients. Even after exclusion of the G-CIMP phenotype, we observed marked differences between the LTS and STS methylome. A total of 1,247 probes in 706 genes were hypermethylated in LTS and 463 probes in 305 genes were found to be hypermethylated in STS patients (p values < 0.05, log2 fold change 6 0.5). We identified 13 differentially methylated regions (DMRs) with a minimum of four differentially methylated probes per gene. Indeed, we were able to validate a subset of these DMRs through a second, independent method (MassARRAY) in our LTS/STS training set (ADCY1, GPC3, LOC283731/ISLR2). These DMRs were further assessed for their prognostic capability in an independent validation cohort (n 5 62) of non-G-CIMP GBMs from the TCGA. Hypermethylation of multiple CpGs mapping to the promoter region of LOC283731 correlated with improved patient outcome (p 5 0.03). The prognostic performance of LOC283731 promoter hypermethylation was confirmed in a third independent study cohort (n 5 89), and was independent of gender, performance (KPS) and MGMT status (p 5 0.0485, HR 5 0.63). Intriguingly, the prediction was most pronounced in younger GBM patients (<60 years). In conclusion, we provide compelling evidence that promoter methylation status of this novel gene is a prognostic biomarker in IDH1 wild-type/non-G-CIMP GBMs.Glioblastoma multiforme (GBM) still constitutes one of the most aggressive and deadly human cancers with a median overall survival (OS) of about 15 months.1,2 Despite this unfavorable prognosis, 16% of GBM patients survive for >36 months.3 These long-term survivors (LTS) usually are younger in age and were shown to have a higher preoperative Karnofsky performance score (KPS). [4][5][6] Recently, a considerable fraction of GBM LTS (34%) were found to harbor IDH1 mutations. 7 Concerted efforts in deciphering the biology of IDH1-mutant GBM have led to the conclusion that these tumors constitute a subgroup differing significantly from IDH1 wild-type GBMs. [8][9][10][11] Gain-of-function mutations